Bacterial aromatic polyketide synthases (PKSs) are a sub-class of PKSs responsible for the biosynthesis of natural products such as doxorubicin and tetracycline. They are composed of 3-10 distinct subunits, which together synthesize a polyfunctional aromatic product. The modularity of these systems has been exploited for the biosynthesis of numerous "unnatural" natural products. Within the past 4 years, we have: (i) Solved the structures of nearly all representative subunits of aromatic PKSs, including the chain initiation ketosynthase (KS), the heterodimeric KS-CLF responsible for chain elongation, the acyl carrier protein (ACP), the malonyl-CoA:ACP transacylase (MAT), and the ketoreductase (KR) from the initiation module;(ii) Dissected the active sites as well as the ACP binding sites of the KS and MAT proteins;iii) Demonstrated that the chain length factor (CLF) does indeed control polyketide chain length;(iv) Identified a new component of a Type II PKS, an acyl-ACP thioesterase;(v) Decoded a highly versatile bimodular mechanism of non-acetate priming of aromatic polyketides;and (vi) Shown that initiation and elongation modules of a bimodular PKS can be predictably recombined to engineer regioselectively modified aromatic polyketides. In the process, several new polyketide compounds were isolated and characterized, including a few with novel biological activities. During the next proposal period, we propose to investigate in greater detail the enzymes responsible for chain initiation, chain elongation and chain termination, and to use these insights to engineer initiation and elongation modules with properties that have not been observed in nature to date. We also propose to test our emerging capabilities for biosynthetic engineering of this class of natural products in the context of two biologically active substances: (i).benzoisochromanequinones as anti-parasitic compounds;and (ii) A- 74528a analogues as 2'-phosphodiesterase inhibitors. Finally, if time permits, we will capitalize on our recent crystal structure of a type I PKS module to develop a fundamentally new strategy for expressing type II PKSs in E. coli, a major unmet need for fundamental and practical biosynthetic objectives.